Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling
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ORIGINAL ARTICLE
Identification through fine mapping and verification using CRISPR/ Cas9‑targeted mutagenesis for a minor QTL controlling grain weight in rice Aye Nyein Chan1,3 · Lin‑Lin Wang1,2 · Yu‑Jun Zhu1 · Ye‑Yang Fan1 · Jie‑Yun Zhuang1 · Zhen‑Hua Zhang1 Received: 25 July 2020 / Accepted: 29 September 2020 © The Author(s) 2020
Abstract Key message A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect. Abstract The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)3/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9–2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8–9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.
Introduction
Communicated by Kan Wang. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00122-020-03699-6) contains supplementary material, which is available to authorized users. * Jie‑Yun Zhuang [email protected] * Zhen‑Hua Zhang [email protected] 1
State Key Laboratory of Rice Biology and Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China
2
Lishui Institute of Agricultural and Forestry Sciences, Lishui 323000, China
3
Advanced Center for Agricultural Research and Education, Yezin Agricultural University, Naypyitaw 15013, Myanmar
Rice (Oryza sativa L.) is one of the most important cereal crops. More than half of the global population use rice as the main food. Development of superior rice varieties is essential to ensure food security. Genetic variation is the basic resource for crop improvement, which has been greatly reduced during domestication and artificial selection (Huang et al. 2012; Wang et al. 2014). Low level of genetic diversity has become a major bottleneck for rice improvement
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